The Nac2 gene of Chlamydomonas encodes a chloroplast TPR-like protein involved in psbD mRNA stability

Abstract

The psbD mRNA, which encodes the D2 reaction center polypeptide of photosystem II, is one of the most abundant chloroplast mRNAs. We have used genomic complementation to isolate the nuclear Nac2 gene, which is required for the stable accumulation of the psbD mRNA in Chlamydomonas reinhardtii. Nac2 encodes a hydrophilic polypeptide of 1385 amino acids with nine tetratricopeptide-like repeats (TPRs) in its C-terminal half. Cell fractionation studies indicate that the Nac2 protein is localized in the stromal compartment of the chloroplast. It is part of a high molecular weight complex that is associated with non-polysomal RNA. Change of a conserved alanine residue of the fourth TPR motif by site-directed mutagenesis leads to aggregation of Nac2 protein and completely abrogates its function, indicating that this TPR is important for proper folding of the protein and for psbD mRNA stability, processing and/or translation.

Specific sequence elements in the 5′ untranslated regions of rbcL and atpB gene mRNAs stabilize transcripts in the chloroplast of Chlamydomonas reinhardtii

“…Light/dark regulation of GUS transcript levels in Chlamydomonas chloroplast transformants carrying rbc L 59 end:GUS genes+ Transformants were grown in 12-h light/12-h dark cycles, total RNA was isolated at time points 11 h dark and 1 h light, and processed as described (legend to Fig+ 2; Materials and Methods)+ RNA gel blots were first hybridized to the atpB probe as a control and, after stripping of the membrane, to the GUS probe+ The increase upon illumination seen in abundance of transcripts of the endogenous atpB gene is due to increased transcription of the atpB gene in light and has been reported previously (Salvador et al+, 1993a)+ The membrane was exposed to X-ray film for 4 h for detection of atpB gene transcripts and for 24 h for detection of chimeric rbc L 59 end:GUS gene transcripts+ Numbers above the lanes denote nucleotide replacements in the rbc L 59 UTR sequence as depicted in Figures 1 and 2+ D: dark (also marked by a filled bar); L: light (also marked by an open bar)+ isolated by genomic complementation (Boudreau et al+, 2000)+ Putative cis-acting elements that are potential targets for RNA-stabilizing proteins have been localized in the 59 UTRs of chloroplast gene transcripts (Higgs et al+, 1999;Nickelsen et al+, 1999)+ A number of proteins were found in in vitro assays to bind to the 59 UTRs of Chlamydomonas chloroplast transcripts (Hauser et al+, 1996) and, in spinach, to the 59 UTRs of transcripts of genes that code for subunits of the ATP synthase complex (Hotchkiss & Hollingsworth, 1999)+ Although the identities and functions of these proteins have not been examined, it is possible that some of them are nucleus-encoded factors involved in mRNA stabilization+…”

Beyond Complementation. Map-Based Cloning in Chlamydomonas reinhardtii

“…Gumpel et al, 1995;Boudreau et al, 2000;Vaistij et al, 2000;Auchincloss et al, 2002;Dauvillee et al, 2003). However, in cases where PS1 suppressors have been recovered from PS2 strains or where a trait otherwise cannot be selected on minimal medium, recovery of the mutation requires other methods.…”

Chloroplast RNA-binding proteins

“…Once RBP40-psbD mRNA complex formation has been established, Nac2 is released from the RNA. This transient interaction of the Nac2 complex with the psbD 5¢UTR is supported by the finding that Nac2 cannot be identified at polysomes from C. reinhardtii chloroplasts (Boudreau et al 2000). Afterwards, RBP40-mediated ribosomal assembly takes place and translation is initiated.…”

“…To date, three nuclear genes have been cloned from C. reinhardtii which encode proteins mediating their function via chloroplast 5¢UTRs. These include Nac2 and Mbb1, controlling the stabilization of psbD and psbB transcripts, respectively, and Tbc2, regulating translation initiation on the psbC mRNA (Boudreau et al 2000;Vaistij et al 2000b;Auchincloss et al 2002). Similar to the above-mentioned genetically defined factors involved in chloroplast-splicing, all three factors have been detected in highmolecular-weight complexes which also contain RNA; but it remains to be clarified whether or not they directly interact with their cognate transcripts.…”

“…Possibly, one of the most comprehensive analyses of this kind was performed for the chloroplast psbD gene, encoding the D2 protein of the PSII reaction center. Analysis of the photosynthetic mutant nac2 revealed that the stability of the psbD mRNA depends on a nucleus-encoded tetratricopeptide repeats protein, which is part of a highmolecular-weight complex mediating its function via the psbD 5¢UTR (Boudreau et al 2000). However, a thorough site-directed mutagenesis of the psbD 5¢region after biolistic transformation of chloroplasts from wild-type cells identified several essential cis-acting elements required for either stabilization or translation of the psbD message (Nickelsen et al 1999).…”